Recent advances in recombinant DNA and genetic techniques have made it possible to introduce and express a desired sequence or gene in a recipient animal. Through the use of such methods, animals have been engineered to carry non-naturally occurring sequences or genes, that is, sequences or genes that are not normally or naturally present in the unaltered animal. The techniques have also been used to produce animals which exhibit altered expression of naturally present sequences or genes.
Animals produced through the use of these methods can be either “chimeric”, in which only some of the animal's cells contain and express the introduced sequence or gene, or “transgenic”, in which all of the cells of the animal contain the introduced sequence or gene. Consequently, in the case of transgenic animals every animal is capable of transmitting the introduced genetic material to its progeny as compared to the chimeric animals in which transmittal to progeny is dependent upon whether the introduced material is present in the germ cells of the animal.
The high efficiency transformation of cultured mammalian cells has been accomplished by direct microinjection of specific DNA sequences into the cell nucleus (Capecchi, M., Cell 22:479-488 (1980)). More specifically, it has also been demonstrated that DNA could be microinjected into mouse embryos and found in the resultant offspring (Gordon et al., P.N.A.S. U.S.A. 77:7380-7384 (1978)). Thus, the ability to produce certain transgenic mice is described and well known.
The basic procedure for producing transgenic mice requires the recovery of fertilized eggs from newly mated female mice and then microinjecting into the male pronucleus of said egg the DNA that contains the sequence or gene to be transferred into the mouse. The microinjected eggs are then implanted in the oviducts of one-day pseudopregnant foster mothers and allowed to proceed to term. The newborn mice are then tested for the presence of the microinjected DNA by means known in the art and appropriate to detect the presence of the microinjected DNA. See, for example, Wagner et al. P.N.A.S. U.S.A. 78:6376-6380 (1981), U.S. Pat. No. 4,873,191, which describes the production of mice capable of expressing rabbit beta-globin in its erythrocytes.
The insulin-like growth factor I receptor (referred to as “IGF-1R” or “IGF1R”) is a transmembrane tyrosine kinase which is overexpressed or activated in many human cancers including colon, breast, lung, prostate, glioblastoma and melanoma. IGF1R is thought to be required for the establishment and maintenance of the transformed phenotype and to exert an anti-apoptotic effect in tumorigenesis. Baserga, R., Cancer Research 55:249-252 (1995). Activation of the receptor is initiated by the binding of either IGF-I or IGF-II to the two α-subunits of the IGF1R, i.e., receptor dimerization, resulting in the autophosphorylation of critical tyrosine residues in the catalytic domain of the β-subunit. Kato et al., J. Biol. Chem. 265:2655-2661 (1993); Gronborg et al., J. Biol. Chem. 258:23435-23440 (1993). Ligand binding also leads to phosphorylation and subsequent activation of downstream substrates thought to be involved in growth regulation and differentiation. Ottensmeyer et al., Biochemistry 39: 12103-12112 (2000). In vitro models have been developed to study the role of kinase dimerization in the activation of the IGF1R that utilize fusion proteins comprising the soluble kinase domain of IGF1R and the homodimeric glutathione S-transferase (GST), Baer et al., Biochemistry 40:14268-14278 (2001).
To date, the transgenic models established for studying IGF1R signaling have been limited to overexpression of the ligands for IGF1 R (Bol et al., Oncogene 14:1725-1734 (1997); DiGiovanni et al., Cancer Research 60:1561-1570 (2000)). These models are further complicated by the existence of a family of IGF binding proteins (IGFBPs), which serve to modulate the bioavailability of free IGF-I and IGF-II, resulting in a long latent period for tumor development.
One approach to studying in vitro the constitutive stimulation of a receptor in the absence of ligand binding activation has utilized a chimeric oncogenic receptor-type tyrosine kinase comprising the extracellular and transmembrane domains of CD8 with the kinase domain of c-Eyk (Zong et al., EMBO Journal, Vol.15, No. 17, 4516-4525 (1996)). The resulting chimera showed elevated kinase activity and caused cellular transformation. Such chimeric receptors may incorporate the extracellular, transmembrane and cytoplasmic domains from the same or different species. The preparation and use of chimeric receptors comprising cytokines and other kinase domains are known in the art. See, for example, Lawson et al., WO 99/57268; M. Roberts, U.S. Pat. No. 5,712,149; and Capon et al., U.S. Pat. No. 5,741,899.
It is therefore of interest to develop a transgenic non-human mammalian model for ligand-independent IGF-1 receptor-driven tumorigenesis. It is also desirable that the animal develops tumors that overexpress IGF1R within a fairly short period of time from birth, facilitating the analysis of multigenerational pedigrees. Such a model can be used to study the pathogenesis and treatment of tumors overexpressing or having activated IGF1R. The instant invention represents such a model using a chimeric receptor.